The very significant potential of using post-column reactors to improve detection in modern liquid chromatography (HPLC) has long been recognized, but little applied. For example, in the publication by Gfeller et al., "Post-Column Derivatization in High-Performance Liquid Chromatography Using the Air Segmentation Principle: Application to Digitalis Glycosides," Journal of Chromatography 142 (1977), pp. 271-281, the authors state:
"Although the use of such reaction techniques after column chromatographic separation has been known for more than a decade with classical column techniques (e.g., amino acid analyzers), little has appeared in relation to modern HPLC. One reason is the many technical problems [.] that still have to be solved." PA0 "The adaptation of reaction detection to modern LC columns requires careful attention to [.] the design of equipment, because extra column effects can be serious. For these reasons reaction detectors have so far found rather limited use in modern LC."
Among specific difficulties that are described in the literature are problems directly involved with state of the the art post-column reactor designs. Thus, Synder et al., "Introduction to Modern Liquid Chromatography", 2nd Ed. (1979), p. 740, state:
Equivalent conclusions are also expressed by Frei et al., "Reaction Detectors in HPLC", Journal of Chromatographic Science, Vol. 17, March 1979, pp. 152-159, wherein the authors state: "the construction of proper reaction detectors comprises a constant struggle against band broadening." In still another recent publication by Jupille, "UV--Visible Absorption Derivatization in Liquid Chromatography", Journal of Chromatographic Science, Vol. 17, March, 1979, pp. 160-167, the author listed among disadvantages of the design or state of the reactors: "a need for hardware modification (with attendant loss in flexibility); and [ ] a risk of band broadening due to post-column mixing volume resulting in loss of resolution."
By way of further explanation, the often mentioned problem of avoiding band spreading is interrelated to various factors, among which is the mode for metering reagent. Any lack in consistency of metering produces fluctuations in reagent concentration in the effluent, which shows up as "noise" in the chromatograph developed by the detector.
The problem is especially severe where highly concentrated reagent is used, since minute fluctuations can produce high background "noise" levels that severely hamper the sensitivity of detection. While one of the choices of the prior art is to use concentrated reagent to avoid band spreading by sample dilution, the gain may, nevertheless, be offset at least partially by increased background noise levels.
Band spreading is also caused by diffusion of sample bands into one another as a function of time. Prior devices inherently appear to obtain poor reagent/effluent mixing, hence, extending the time factor. The deficiency is particularly shown by the description in the literature of reagent/effluent mixing devices as means for promoting faster reactions.
Prior solutions to these and other related problems can thus be said to often involve serious drawbacks, e.g., increased "noise", or may be equally as objectionable on the basis of adding too much complexity, e.g., air segmentation methods, to apparatus which has been characterized as already involving inherently disadvantageous "hardware modifications" and "attendant loss of flexibility".
An objective of the invention is accordingly to provide an improved liquid chromatographic method and apparatus characterized by the development of an improved post-column reactor which requires little in the way of hardware modifications.
It is particularly an objective hereof to provide such improved method and apparatus which achieve essentially constant "pulseless" metering of reagent, and, in addition, significantly improved diffusion of the reagent into the chromatographic column effluent.
It is still a further objective hereof to provide such apparatus and method wherein sample dilution is minimized without need for resorting to the addition of highly concentrated forms of reagent as a means or requirement, or resorting to other objectionable process or apparatus restrictions.
Still a further objective hereof is to provide such method and apparatus, which in contrast with prior art methods and apparatus, are of significantly less cost to use and to maintain.